Proteasome-mediated protein degradation is a central process in the regulation of cell function that usually requires prior substrate modification by a multi-ubiquitintag. Ornithine decarboxylase (ODC), unusually, does not require prior ubiquitin modification for proteasome degradation, and has been the chief focus of our studies. Mammalian ODC participates in a unique form of feedback regulation, whereby polyamines promote its destruction by inducing the protein antizyme 1 (AZ1). ODC and AZ1 interact, enhancing ODC destruction by the proteasome. During the previous funding period we made significant findings and established technical advances that will provide the basis for further progress. Each of the Specific Aims below seeks to answer an important question about the proteasome and ODC degradation. Specific Aim 1. What are the salient structural features in ODC and AZ that are needed for proteasomal degradation? How do these interact? The functions of ODC cysteine 441 will be probed by mutation and by determining whether it undergoes chemical modification. AZ1 residues 131 and 145 will be examined by introducing crosslinkers within the region that they span and examining products of cross-linking to ODC. The structure of ODC will be probed by X-ray crystallography and NMR to determine the conformational consequences of its interaction with AZ1. Specific Aim 2. What interactions of the ODC-AZ complex with the proteasome are required for ODC degradation? Do these interactions differ for ODC-AZ and ubiquitinated substrates of the proteasome? Contacts between substrates and the proteasome from initial binding to translocation will be mapped. Stop-transfer sequences will be introduced into substrates and their effect on insertion and degradation determined. Structurally simplified ("lidless") and mutant proteasomes (ATPase homolog substitutions)will be utilized to determine the functional role of proteasome proteins and subassemblies. Specific Aim 3. What cellular components participate in and modulate substrate recognition and proteasomal activity? Genetic screens will be preformed to identify these. Genetic screens will be carried out in the budding yeast Saccharomyces cerevisiae using mouse ODC and derived reporter/selection constructs to identify gene products that influence ODC degradation.